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PLoS One. 2019 Jan 15;14(1):e0208237. doi: 10.1371/journal.pone.0208237. eCollection 2019.

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells.

Author information

1
Innovative Genomics Institute, University of California, Berkeley, CA, United States of America.
2
Children's Hospital Oakland Research Institute, UCSF Benioff Children's Hospital, Oakland, CA, United States of America.
3
Research Programs Unit, Molecular Neurology and Biomedicum Stem Cell Centre, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
4
Clinical Genetics, HUSLAB, Helsinki University Central Hospital, Helsinki, Finland.
5
Blood and Marrow Transplant Program, Division of Hematology, UCSF Benioff Children's Hospital, Oakland, CA, United States of America.
6
Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.
7
Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.
8
Department of Molecular and Cellular Biology, University of California, Berkeley, CA, United States of America.

Abstract

Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as 𝛾-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential 𝛾-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated 𝛾-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases in 𝛾-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of 𝛾-globin. These data suggest that the 1.7 kb region is not an autonomous 𝛾-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of ß-hemoglobinopathies.

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